PROJECT SUMMARY The pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that coordinates the dynamic assembly of complexes essential for the function of pancreatic ?-cells. Disruption of these complexes contributes to the molecular basis for type 2 or maturity onset diabetes of the young (MODY); related to its role in development, dysregulation of Pdx1 is associated with pancreatic cancer. Mechanistic studies of this essential protein have not kept up with advances in understanding of its central role in pancreatic health. It is the central hypothesis of this proposal that the unique structural features of binding motifs in the Pdx1 transactivation domains, and their modulation by post-translational modification, play an essential role in regulating the strength of Pdx1's protein-protein interactions. This project presents a unique opportunity to have a sustained impact in this challenging area because it leverages an innovative 13C direct-detect NMR strategy custom-designed in the PI?s laboratory to probe the biophysics of transcription factors and the complexes they form. In this context, the first specific aim of this project is to characterize the role of the Pdx1 C-terminal domain in linking Pdx1 stability to glucose sensing, through interactions with the ubiquitin ligase adaptor protein SPOP. This aim tests the working hypothesis is that multivalent Pdx1-SPOP interactions provide the avidity required to promote Pdx1 turnover, whereas the weak interactions mediated by individual SPOP binding motifs would fail. This predicts that loss of individual interactions through disease-driving mutations, or clinical intervention, is sufficient to prevent Pdx1- SPOP interactions. NMR and crystallography will provide structural insights into the SPOP recruitment mechanism, while binding assays further test the multivalence model. These biophysical studies will be connected to functional assays in cultured cells to establish that the molecular details uncovered drive phenotypes. The second specific aim is to evaluate the mechanism whereby Pdx1 recruits the co-activator Set7 to protein complexes. This aim?s working hypothesis is that Pdx1-Set7 interactions are mediated by the N- terminal domain of Pdx1 and the Set7 N-Set domain. Binding assays and NMR studies will develop structure- function relationships that will be validated in cells. The third specific aim is to investigate the regulation of Pdx1 function through post-translational modifications. Phosphorylation and methylation establish temporal control over Pdx1 interaction specificity and half-life. This aim?s working hypothesis is that phosphorylation modifies the strength of Pdx1 interactions with both SPOP and Set7. Separately, methylation of Pdx1 by Set7 stimulates Pdx1 activity. Thus, it is also the working hypothesis of this aim that methylation of Pdx1, not direct histone methylation, is responsible for gene regulation by Set7 in ?-cells. The proteins Pdx1, SPOP, and Set7 are all known contributors to diabetes, as well as to the etiology of adenocarcinomas with broad primary tissue distributions. Findings from this project are expected to generalize to all protein-protein interactions involving SPOP, the family of SET paralogs, and the transcription factors that interact with them in a range of significant biomedical contexts.